Abstract: Growing concern over bacterial food contamination has led to increased examination of food testing protocols in today’s industry. Currently, the use of bacterial strains as positive controls in testing protocols is not widely practiced for fear of cross-contaminating samples. Due to ongoing scrutiny of food testing methodology and growing regulations under the Food and Drug Administration (FDA) Food Safety Modernization Act, it is imperative to have control strains with unique, easily detectable traits that distinguish positive control strains from actual food contaminants, diminishing the fear of cross-contamination and improving current practices. In this study, we developed GFP reporter-labeled Shiga toxin-producing Escherichia coli for use as controls in QC testing.

Abstract: Herpes simplex virus (HSV-1 and HSV-2) causes a wide range of clinical manifestations that result in lifelong infections. Quantitative PCR (qPCR) assays are routinely used for the detection of HSV-1 and HSV-2 infections in clinical samples. However, the accuracy of a qPCR assay is dependent upon the generation of a standard curve using a positive control with a known genome copy number. Moreover, an independent positive control is required to monitor variations in assay performance for molecular assays. ATCC has developed HSV-1 and HSV-2 quantitative molecular standards for use as controls for the detection and quantification of these viruses from clinical samples.

Poster presented at the 26th European Congress of Clinical Microbiology and Infectious Disease, April 2016.

Abstract: Viral hepatitis caused by hepatitis B virus (HBV) and hepatitis C virus (HCV) is a major health concern and affects millions of people worldwide. Patients are routinely monitored by quantitative RT-PCR (qRT-PCR) for the presence of HCV RNA or by qPCR for HBV DNA in blood. Since these viruses are difficult to culture in vitro, obtaining control material for these molecular-based assays is a challenge. To address this problem, ATCC has developed HBV and HCV specific quantitative synthetic molecular standards for use as controls for the detection and quantification of these viruses from clinical samples.

Abstract:In vivo studies have shown that kidney membrane transporters play a key part in drug disposition and renal clearance. One such transporter is OAT1 (SLC22A6), which is critical for maintaining homeostasis of endogenous substances. This makes OAT1 a good transporter to assay for drug interactions with the kidney. Unfortunately, primary cells lose OAT1 expression in culture, and transiently expressed OAT1 has great variations between production lots, which make data hard to interpret. In our study we have generated HEK 293T/17 cells that stably overexpress the OAT1 gene driven by the human elongation factor-1 alpha (EF1α) promoter. After confirming the mRNA expression by RT-PCR, we performed immunostaining that indicated OAT1 is correctly trafficked to the membrane. Most importantly, we validated that the overexpressed OAT1 transporter has normal transport activities by using 5-carboxyfluorescein (5-CF) and para-aminohipurate (PAH; data not shown) uptake assays, and that the uptake can be inhibited by the well-known inhibitors probenecid and novobiocin. Both inhibitors responded in a dose dependent manner for 5-CF uptake with IC50 values between 5-16 μM. Even at higher passages, the cell line retained the functionality of OAT1. Overall, our data has shown that this modified cell line is a very useful in vitro tool for testing regulation of OAT1 membrane transporter activity in kidney cells.

Abstract:Recent studies show that tumor cells derived from a subset of patients with non-small-cell lung cancer (NSCLC) harbor the echinoderm microtubule-associated protein-like 4 (EML4)-anaplastic lymphoma kinase (ALK) fusion oncogene; the result of a Paracentric chromosomal inversion on the short arm of chromosome 2. The EML4-ALK oncogene, like other ALK fusion oncogenes, is a druggable target that is responsive to ALK inhibitors. However, there is a lack of EML4-ALK in vitro models for drug screening. Here we set out to generate an isogenic EML4-ALK fusion non-small cell lung cancer model in the A549 lung cancer cell line, which harbors other naturally occurring genomic aberrations inherent in non-small cell lung cancer. This model could serve as a clinically relevant drug screening cell model. In this study, we utilized the CRISPR/Cas9 genome editing platform to target endogenous loci in human cells and create the intended genomic translocation event. By employing sgRNAs-Cas9 constructs designed to cut precisely at relevant translocation breakpoints, we induced cancer-relevant genomic rearrangements that resulted in the expression of EML4–ALK fusions. Breakpoint junction analysis tested after sgRNA-CRISPR/Cas9 mediated genomic DNA cleavage in A549 cells showed the successful creation of the EML4-ALK fusion found in tumor cells from a subpopulation of NSCLC patients. Furthermore, single clonal isolation and functional screening demonstrated that the EML4-ALK isogenic cell line was sensitive to ALK inhibitors relative to the parental A549 cell line. This newly developed EML4-ALK isogenic lung cancer cell line could provide a very useful tool for oncology drug discovery and development.

Abstract:This study will suggest that mesenchymal stem cells (MSC) with their inherent multi-potency are a better alternative to fibroblasts in certain co-culturing situations. Cell-based assays that use primary cells do offer better predictability, but they are hampered by the finite life span of primary cells, which causes inconsistency in the results from donor variation. hTERT-immortalized cell lines solve this problem by offering the functionality of a primary cell with the longevity of a cell line. In this study we characterize the functionality of an hTERT immortalized adipose tissue-derived MSC cell line (hTERT-MSC) and illustrate its use in two co-culturing applications: wound healing and angiogenesis We confirmed that primary keratinocytes and an hTERT-immortalized keratinocyte cell line, Ker-CT, are able to fully differentiate into skin equivalents in an air-liquid interface (ALI) 3D culture model, when co-cultured with hTERT-MSCs. To confirm the functionality of the co-culture models, both primary keratinocytes and Ker-CT ALI co-cultures were subjected to a scratch assay. Re-epithelialization occurred in both cell lines. Additionally, we established an in vitro angiogenesis co-culture model system using TeloHAEC-GFP (an immortalized aortic endothelial reporter cell line stably expressing GFP), and hTERT-MSCs. Results show that the new model can form tubular structures in less than 7 days instead of 14 days compared to co-culture with fibroblasts, and also responds effectively to VEGF stimulation and drug treatments. Furthermore, immunofluorescence staining shows that cells surrounding the tubular structures stain positive for αSMA supporting the physiological relevance of this in vitro model system. The co-culture models developed by using hTERT-MSCs in this report provide a more consistent and robust in vitro co-culture system for studying wound healing and vascular biology for drug screening and tissue engineering

Abstract: Human induced pluripotent stem cells (iPSCs) have the capacity to differentiate into all of the somatic cells types and hold great promise for both regenerative medicine and drug discovery. A need for better tools to address neurological disease modeling and neuro-toxicology screening exists. We have developed a scalable process that allows for the generation of large quantities of neural progenitor cells (NPCs) derived from normal and Parkinson’s disease iPSC lines, along with a serum-free defined NPC expansion medium and dopaminergic differentiation medium.
To validate the process of NPC derivation, we generated NPCs derived from iPSCs that were reprogrammed with the Sendai virus from the following sources: human foreskin fibroblasts (HFF-1), human CD34+ cells, and human fibroblasts from a patient with Parkinson’s disease. Compared to Parkinson’s disease patient-derived NPCs, both normal NPC lines demonstrated greater proliferative capacity. Moreover, our CD34+ cell-derived NPCs possessed better tri-lineage differentiation efficiency than that of fibroblast-derived NPCs although all three types of NPCs were capable of differentiating into dopaminergic neurons, astrocytes, and oligodendrocytes.

Abstract: Noroviruses (NoV) are the most common cause of epidemic gastroenteritis, accounting for 95% of viral gastroenteritis outbreaks worldwide. NoV detection is difficult because they are genetically heterogeneous and cannot be grown in cell culture. The principle detection method utilized by diagnostic laboratories is quantitative RT-PCR (qRT-PCR). The accuracy of a qRT-PCR assay relies on the generation of a standard curve using a positive control with a known genome copy number. To that end, we have developed quantitative synthetic standards that include conserved sequences from NoV GI and NoV GII for the detection and quantification of NoV from either clinical, food, or environmental samples. These quantitative synthetic molecular standards provide well-characterized reference materials for the detection and quantification of NoV by qRT-PCR. Further, they exhibit excellent compatibility with numerous published NoV assays and can be used as controls for assay development, verification, and validation.

Abstract: In this study, we compared primary keratinocytes (ATCC® No. PCS-200-010) to hTERT immortalized keratinocytes (Ker-CT; ATCC® No. CRL-4048™), co-cultured with either primary fibroblasts (ATCC® No. PCS-201-010), primary adipose-derived mesenchymal stem cells (MSCs; ATCC® No. PCS-500-011), hTERT-immortalized fibroblasts (BJ-5ta; ATCC® No. CRL-4001™), or hTERT-immortalized MSCs (hTERT-MSCs; ATCC® No. SCRC-4000™). We confirmed that both primary keratinocytes and Ker-CT are able to fully differentiate into skin equivalents in a 3D culture model when co-cultured with primary fibroblasts, primary MSCs, BJ-5ta, or hTERT-MSCs. To confirm the functionality of the co-culture models, both the primary keratinocytes and the Ker-CT air-liquid interface (ALI) co-cultures were subjected to a scratch assay. Re-epithelialization occurred in both cell lines, and interleukin 8 (IL-8) showed an increase in expression from day 0 to day 1 and 3, corresponding to migration of cells into the wound. The continuous nature of the Ker-CT cell line makes it an invaluable model for the research of keratinocyte biology, as it eliminates the issue of short life span and donor variation seen with primary cells.

Abstract: In this study, we established an in vitro co-culture model system using the aortic endothelial cell line TeloHAEC (hTERT immortalized human aortic endothelial cell line, ATCC® No. CRL-4052™) and the hTERT immortalized adipose-derived mesenchymal stem cell line hTERT-MSC (ATCC® No. SCRC-4000™). Both cell lines were immortalized by hTERT (human telomerase reverse transcriptase) alone and have been well-characterized showing that the cells retain the most important characteristic of their parental counterparts; TeloHAECs co-cultured with BJ primary fibroblasts (ATCC® No. CRL-2522™) for 14 days in optimized ATCC angiogenesis medium formed fine tubular structures as shown by staining with CD31 endothelial cell marker. The tubule length elongated with increasing doses of vascular endothelial growth factor (VEGF), and tubule formation can be completely blocked by suramin in a concentration-dependent manner. Next, we introduced GFP into the TeloHAEC cell line (TeloHAEC-GFP, ATCC® No. CRL-4054™), allowing for real time visualization of angiogenesis when co-cultured with BJ fibroblasts; another hTERT immortalized cell line, hTERT-MSC, replaced the BJ-primary fibroblast in the co-culturing system. It was observed that the new model forms the tubular structures in less than 7 days, and also responds effectively to VEGF and compounds such as suramin. Further, the hTERT-MSC cells which surround the tubular structures have undergone transformation indicated by positive αSMA staining (a marker of smooth muscle cells), this indicates that the system has physiological relevance. Therefore, the co-culture models developed by using hTERT-immortalized cell lines described in this report provide a consistent and robust in vitro system for studying vascular biology, drug screening and tissue engineering.

Poster presented at the Society for Neuroscience Meeting, November 2014.

Abstract: Dopaminergic neurons play significant roles in motor, reward, and motivational behavior related circuits throughout the brain. To date, there are few continuous in vitro models available to laboratories in research, industry, and academia for studies related to basic dopaminergic cell biology or high throughput screening. Here, we propose the use of a human model system, LUHMES cells (ATCC® CRL-2927™), to study dopaminergic neuron cell biology and Parkinson’s disease. LUHMES cells are neuronal precursors derived from fetal ventral mesencephalon. Neuronal differentiation is governed by the termination of v-myc expression using low levels of tetracycline. During our characterization, we found that tetracycline induced differentiation resulted in consistent neurite outgrowth in LUHMES cells within two to four hours. One day post differentiation, cells displayed similar morphology, with several long processes protruding from the cell soma. Growth cones were often observed in early differentiated cultures. Immunocytochemistry in early differentiated cultures (Days in vitro, DIV 2-3) revealed low level expression of tyrosine hydroxylase; however, these levels were increased significantly by 7 DIV with many neurons expressing tyrosine hydroxylase. We also investigated dopamine transporter expression. Differentiated LUHMES cultures were positive for neuronal markers such as bIII tubulin and devoid of expression of traditional glial markers including GFAP and IBA-1. Both undifferentiated and differentiated LUHMES cells were easily transfected using basic eGFP constructs, although greater efficiencies were observed with the use of viral constructs. In summary, LUHMES cells are a suitable and robust in vitro model system for studying dopaminergic neuron cell biology and mechanisms underlying Parkinson’s disease.

Abstract: Large-scale cancer genome programs have generated a rich data set comprising genetic abnormalities observed in thousands of clinical patient tumors, which provides a major opportunity for the molecular detection of cancer. However, the lack of controls for molecular tests has been a challenge. Because of the reproducible nature of the cell lines, genomic DNAs of fully characterized and authenticated cell lines provide a solution.

Genomic DNAs were extracted from over 70 commonly used human cancer cell lines derived from the breast, lung, colon, and pancreas, as well as hematopoietic and lymphoid tissue. Cancer gene mutations were identified by next-generation sequencing. Gene copy number changes were analyzed using the qBiomarker Copy Number PCR Assays kit (QIAGEN). Moreover, the selected cell lines were analyzed by quantitative polymerase chain reaction (qPCR), Western blot, and immunofluorescence (IF) staining to verify gene and protein expression mutation.

Abstract: Positive controls are essential for establishing assay performance and equipment efficacy. Yet, some food testing laboratories refrain from using bacterial strains as positive controls for fear of cross-contaminating their samples. Under the Food and Drug Administration (FDA) Food Safety Modernization Act, laboratories face an increasing number of regulations to expand testing for objectionable organisms. Control strains with unique, easily detectable traits which distinguish positive control strains from actual food contaminants can help differentiate true contamination from control strain cross-contamination.

In this study, we introduced shuttle vectors encoding either green fluorescent protein (GFP, Life Technologies) or NanoLuc® (Promega) into Escherichia coli strains, including Shiga toxin-producing O157 (stx1+, stx2+, eaeA+) and non-Shiga toxin-producing O157 (stx1-, stx2-, eaeA-), for use in food pathogen detection assays. Both reporters can be easily visualized without specialized detection equipment; GFP fluoresces when excited by UV light, while bacteria engineered with NanoLuc emit a strong light signal in the presence of a chemical substrate. Upon establishing the detectability of NanoLuc in the E. coli O157 strains, the reporter was transformed into the “Big Six” non-O157 E. coli strains (serogroups: O26, O45, O103, O111, O121, and O145) for use as reporter-labeled positive controls.

Abstract: Fluorescent proteins, such as green fluorescent protein (GFP), have diverse applications in the basic and applied sciences. While GFP has been frequently used in eukaryotic systems, its applications have been limited in microorganisms due to a lack of broad-range molecular tools. In this study, we have developed a vector to express GFP in pathogenic bacteria for use in bacterial pathogenesis and pathogen-host studies. A shuttle vector encoding the GFP variant mut31 (pUCP18-MCSgfpmut3) was generated and successfully transformed into various Gram-negative opportunistic pathogens from the ATCC collection, including: Escherichia coli (ATCC® 25922™), Salmonella enterica (ATCC® 14028™), Shigella flexneri (ATCC® 12022™), Pseudomonas aeruginosa (ATCC® 10145™), and the P. aeruginosa type strain PAO1 (ATCC® 15692™). P. aeruginosa was used as a model to test the characteristics of the vector and sensitivity of detection using a fluorescence plate reader, microscopy, flow cytometry, and in vivo imaging systems.

Abstract: Influenza is one of the most significant causes of acute respiratory infection worldwide. Rapid diagnostic tests for highly contagious pathogens, such as Influenza, are essential for decreasing the public health impact of emerging infectious diseases and bioterrorism agents. However, these tests require positive controls that are not always readily available. Consequently, if worldwide public health laboratories are unable to meet the costly regulations required for the import, transfer, and safe use of pathogens used as controls, then critical diagnostic, surveillance, and epidemiological information could be missed.

The use of in vitro synthesized viral RNA as a control would provide essential equivalency standards that would be accessible to any laboratory performing quantitative RT-PCR tests. Synthetic RNA controls are particularly useful for laboratories which lack appropriate biosafety containment facilities for propagating a particular pathogenic virus, or have difficulty gaining access to the organism in question due to international tightening of both import and export controls.

Abstract: Dengue fever is an acute illness caused by any one of four serotypes (1-4) of genetically related dengue viruses (DENV), with an estimated 390 million cases reported annually. Currently, quantitative RT-PCR (qRT-PCR) is the preferred method for the detection and quantification of DENV in clinical diagnostics and epidemiological surveillance. The accuracy of a qRT-PCR assay relies on the generation of a standard curve using a positive control with a known viral genome concentration.

Native DENV RNA can be used as a standard for these assays; however, the full-length dengue viral RNA is on the Commerce Control List and requires a permit from the US Department of Commerce for international shipment. To make DENV RNA standards more accessible, ATCC has developed four synthetic molecular standards that represent DENV serotypes 1-4. Each standard contains short fragments from the capsid, membrane, and envelope genes of the DENV genome, as well as target regions encompassing the primer sequences from numerous published RT-PCR assays, including the DENV-1-4 Real-Time RT-PCR Assay developed by the CDC1. The synthetic RNA standards were quantified by Droplet Digital™ PCR (ddPCR™) in order to package precise copies of RNA. Moreover, given the inherent labile nature of RNA, a stabilization matrix was added to the quantitated RNA preparation. As compared to native RNA, these synthetic standards are easier to use as controls for qRT-PCR assays, exhibit less variability, have a longer shelf life, eliminate the need to culture viruses and can be used under BSL-1 conditions. Further, this synthetic quantitative RNA approach can be extended to other pathogenic viruses which are unculturable or need to be grown in a high-containment facility.

In the following proof-of-concept study, we amplified the synthetic molecular standards with the published primers from the CDC assay1 and Waggoner et al2, and used the generated standard curves to quantify viral RNA extracted from various DENV strains.

Abstract: Patient-specific induced pluripotent stem cells (iPSCs) provide a unique tool for the study of human diseases such as Parkinson’s disease. To provide a better research tool for studying Parkinson’s disease, we generated three iPSC lines, from dermal fibroblasts of a 63 year old Caucasian male, diagnosed with Parkinson’s disease, by reprogramming with sendai viral, retroviral, or episomal expression of OCT3/4, SOX2, KLF4, and MYC genes. The Parkinson iPSC lines generated with different reprogramming methods all demonstrated similar cell morphology, pluripotent marker expression, and the ability to differentiate into three germ layers. Compared to an hiPSC line-derived from a healthy subject, these Parkinson’s iPSC lines showed similar efficiency of neural differentiation into neural progenitors from iPSC-derived embryoid bodies. To more effectively model Parkinson disease, we have sequenced all exons of the three Parkinson iPSC lines along with their parent fibroblast by exome sequencing with an Agilent’s SureSelect 51 Mb array. Compared to the hg19 human genome reference, each cell line has over 300 genes with missense mutations and there are 226 genes with missense mutations conserved among all four cell types. More importantly, there are three amino acid changes within the LRRK2 gene, the most common Parkinson’s disease-related gene, at positions 50 (R50H), 723 (I723V), and 2397 (M2397T), which have previously been reported in Parkinson’s patients. Via integrating and non-integrating reprogramming methods, we have created three fully characterized iPSC lines that carry LRRK2 mutations.

Poster presented at the American Association of Cancer Research Annual Meeting in Washington, DC, April 2013.

Abstract: Increased understanding of cancer genome is affecting every corner of cancer research. Although human tumor cell lines have been used as essential tools for decades, there are only a few cell line panels have been developed for the drug screening. There is a gap between the new knowledge of cancer genome and the cell line based platforms for both basic and translational research. Here, we show that new generation tumor cell panels are filling the gap. The panels were generated by selecting authenticated cell lines derived from variant cancer types, and annotated with genetic alteration information generated by large scale sequencing projects such as the Catalog of Somatic Mutations in Cancer (COSMIC) and the Cancer Cell Line Encyclopedia (CCLE). To capture the genetic diversity of cancer, each panel includes cell lines with varying gene mutation complexity. To further facilitate targeted drug discovery, the molecular signature tumor cell line panels focus on individual driver genes, critical protein kinases, transcription factors and cell signaling pathways. Those panels have been analyzed to verify gene mutation, gene expression, protein expression and bio-functions.

ATCC Vendor ShowcasePresentation at ASCB 2013

ATCC Human telomerase (hTERT) immortalized cell lines combine the in vivo nature of primary cells and the long culture life of continuous cell lines. This section will provide an overview of the hTERT-immortalized cell line collection, and will examine the use of RPTEC/TERT1 (ATCC® No. CRL-4031™) and TIME (ATCC® No. CRL-4025™) cell lines to demonstrate how hTERT-immortalized cell lines can help you reach your research goals.

ATCC has a wide selection of induced pluripotent and mesenchymal stem cells along with an array of associated culture media and reagents. This section will provide an overview of the ATCC stem cell collection and describe how these resources can be used in cell biology studies.

ATCC offers a superior lipid-based transfection reagent (TransfeX) that can be used to transfect difficult cell types, like primary and stem cells, and uncomplicated continuous cell lines. In this section, we will show how TransfeX provides higher transfection efficiency and lower cytotoxicity than other commercially available transfection reagents. We will also describe the HEKPlus Expression System for protein expression.

ATCC molecular signature panels focus on key components of cell signaling pathways such as EGFR, AKT, PI3K, PTEN, or p53. This section will describe how we generated these panels using authenticated cell lines containing critical gene copy number changes and site mutations, as well as how each panel was experimentally validated for genetic alterations, protein expression, and cell functionality.